TW202229548A - Tetrameric alpha/beta hydrolase variants with increased temperature stability and methods of using and producing thereof - Google Patents

Abstract

The present invention relates to alpha/beta hydrolase variants with improved properties compared to a parent alpha/beta hydrolase, e.g., having increased temperature stability, methods of using and producing such variants including methods of directing the quaternary structure formation from homo-dimers to homo-tetramers.

Description

Tetrameric alpha/beta hydrolase variants with improved temperature stability and methods for their use and production

The present invention relates to alpha/beta hydrolase variants with improved properties compared to the parental alpha/beta hydrolase of SEQ ID NO: 1, for example having improved temperature stability, and to polynucleosides encoding the variants Acids, methods of using and producing the variants, including methods for directing the formation of quaternary structures from homodimers to homotetramers. In particular, the present invention relates to methods of degrading zearalenone (ZEN) using this variant.

Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative of mycotoxins is zearalenone (ZEN), formerly known as F-2 toxin, produced by a variety of Fusarium species and found throughout the world. These fungi infect cultivated plants, especially such as various grains, where fungal infection usually occurs before harvest, when fungal growth and/or mycotoxin production can occur before storage, or fungal infection can even occur after harvest, Before storage or under inappropriate storage conditions. The United Nations Food and Agriculture Organization (FAO) has estimated that 25% of the world's agricultural products are contaminated with fungi, causing serious economic losses. In an 8-year international study, a total of 19,757 samples were analyzed from January 2004 to December 2011; 72% of them tested positive for at least one fungus, 39% were found to be contaminated, 37% tested positive for ZEN (Schatzmayr and Streit (2013) 'Global occurrence of mycotoxins in the food and feed chain: Facts and figures.' World Mycotoxin Journal 6(3):213-222). ZEN has been found in all regions of the world and in all types of cereal and feed crops tested such as corn, soybean meal, wheat, wheat bran, DDGS (distillers dried grains and solubles) and in finished animal feed mixtures, incidence to 100%.

ZEN binds to estrogen receptors and causes hormonal disturbance, ZEN is absorbed immediately after oral administration and converted by animals to two stereoisomeric metabolites, alpha-zearalenol (alpha-ZEL) and/or beta- Zearalenol (β-ZEL). For example, [alpha]-ZEL, and [alpha]-zearatol ([alpha]-ZAL) and/or zearalenone (ZAN), have stronger estrogenic effects than ZEN. Although the estrogenic activity of conjugated ZEN derivatives is lower than that of ZEN itself, ZEN is re-released from these conjugated ZEN derivatives in the digestive tract, restoring its full estrogenic activity.

ZEN has an oral LD50 of up to 20000 mg/kg body weight, and subacute and/or subchronic toxic effects such as teratogenicity, carcinogenicity, estrogenic effects and immunosuppressive effects can occur in animals or humans with prolonged exposure. Feed contaminated with ZEN can cause developmental disorders in mammals. Pigs, especially piglets, are extremely sensitive to ZEN. Concentrations of ZEN in the feed above 0.5 ppm can cause stunted growth, and concentrations above 1.5 ppm can cause high estrogen in pigs. In cattle, ZEN at 12 ppm caused spontaneous abortion.

Due to the rapid absorption of ZEN through the mucosa (especially through the gastric mucosa and oral mucosa), immediate quantitative inactivation is necessary. ZEN can be detected in blood 30 minutes after oral administration. Due to the harmful effects of ZEN, the European Union has given a maximum limit of ZEN in food and a recommendation for an upper limit of ZEN in animal feed products (EC No. 1881/2006).

The main strategy to reduce ZEN in food and animal feed products is to limit fungal growth, for example by maintaining "good agricultural practices". This includes many measures including keeping seeds free from insects and fungi or removing agricultural waste from fields in a timely manner. In addition, fungal growth in fields can be reduced by the use of fungicides. After harvesting, harvested material should be stored with residual humidity below 15% and low temperature to control fungal growth. Likewise, fungal-infected material should be removed prior to further processing. Despite all these precautions, between 2004 and 2011, even in regions with the highest agricultural standards such as North America and Central Europe, up to 37% of corn samples tested were found to be contaminated with ZEN (Schatzmayr and Streit (2013)).

Another related mycotoxin is ochratoxin A (OTA; also known as N-{[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-di Hydrogen-1H-2-benzopyran-7-yl]carbonyl}-L-phenylalanine, (−)-N-((5-chloro-8-hydroxy-3-methyl-1-oxo (2S)-2-{[(3R)-5-chloro-8-hydroxy-3-methyl-1 -Oxo-3,4-dihydro-1H-2-benzopyran-7-carbonyl]amino}-3-phenylpropionic acid, (R)-N-((5-chloro-3,4- Dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl)phenylalanine, N-(((3R)-5-chloro- 8-Hydroxy-3-methyl-1-oxo-7-isochromanyl)carbonyl)-3-phenyl-L-alanine, N-[(3R)-5-chloro -8-Hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-2-benzopyran-7-carbonyl]-L-phenylalanine, N-{[(3R) -5-Chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isobenzopyran-7-yl]carbonyl}-L-phenylalanine), The CAS number is 303-47-9, which is abundantly produced by fungi and is the most toxic member of the ochratoxin group including ochratoxin B, ochratoxin C, and others. In particular, OTA has received close attention in food and feed safety due to its severe adverse effects in humans and animals, including nephrotoxicity, immunotoxicity, and carcinogenicity (Carballo et al., 2019; Malier et al., 2016). In this regard, the International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen in Group 2B (IARC, 2012).

To address the above problems and deficiencies, there is a need to develop α/β-hydrolase variants that are capable of removing ZEN toxicity at higher temperatures and are suitable for use as food or feed additives or food or feed products.

The present invention has been made in view of the above-mentioned prior art. Accordingly, an object of the present invention is to provide, inter alia, an improved means and method of removing the toxicity of ZEN (eg, to provide, among other things, a composition capable of removing other related mycotoxins).

The present application satisfies this need by providing variations, compositions and methods based on the description below, characterized by the scope of the claims and described by the accompanying examples and figures.

The present invention relates to variants of the parental alpha/beta hydrolase comprising substitutions at one or more positions corresponding to: SEQ ID NO: 1 (the numbering of SEQ ID NO: 1 is preferably used) 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69 , 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304, wherein the variant has alpha/beta hydrolase activity, and wherein the variant is with SEQ ID NO: 1 The polypeptide has at least 71% (preferably: at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% , at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%) but less than 100% sequence identity A polypeptide, the variant has improved temperature stability compared to the parental alpha/beta hydrolase.

The present invention also relates to a method for degrading zearalenone and/or derivatives thereof, comprising: (a) providing one or more of the following: a variant of the present invention and/or a parental alpha/beta hydrolase , preferably the parent alpha/beta hydrolase having at least 95% sequence identity with the polypeptide of SEQ ID NO: 1; (b) making one or more polypeptides of (a) with zearalenone and/or Derivatives thereof are contacted (eg, to form an enzyme-substrate mixture).

The present application satisfies this need by providing the variants and compositions described below, characterized by the scope of the claims, and illustrated by the accompanying examples and figures.

Sequence Listing Overview

SEQ ID NO: 1 is the amino acid sequence of the parental alpha/beta hydrolase.

SEQ ID NOs: 2-35 are variant alpha/beta hydrolase amino acid sequences.

SEQ ID NO: 36 is common amino acid motif 1.

SEQ ID NO: 37 is common amino acid motif 2.

SEQ ID NO: 38 is common amino acid motif 3.

SEQ ID NO: 39 is common amino acid motif 4.

definition

An "EC number" (Enzyme Commission Number) as used herein may be used to refer to enzymatic activity according to the Enzyme Nomenclature Database published on 26 February 2020 (eg, available at https://enzyme.expasy.org/) . EC numbers refer to the NC-IUBMB Enzyme Nomenclature 1992 (Academic Press, San Diego, CA), including Supplements 1-5 published in Eur. J. Biochem. 1994, 223, 1-5, respectively ; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650.

As described herein, reference is made to the UniProtKB accession number (http://www.uniprot.org/, available from UniProtKB version 2020_06 published on 2 December 2020)

The term "polypeptide" is used herein equivalently to the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, typically less than 30 amino acids) comprise one or more amino acids linked to each other by covalent peptide bonds (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules, eg consisting of more than 30 amino acids. Polypeptides can further form multimers, such as dimers, trimers and higher order oligomers, ie, consist of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be the same or different. Accordingly, the corresponding higher order structures of such multimers are referred to as homodimers or heterodimers, homotrimers or heterotrimers, and the like. An example of a heteromultimer is an antibody molecule, which in its naturally occurring form consists of two identical light peptide chains and two identical heavy peptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides/proteins, wherein the modification results from, for example, post-translational modifications (eg, glycosylation, acetylation, phosphorylation, etc.). Such modifications are known in the art.

Sequence identity : The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) was used to determine the sequence identity between two amino acid sequences, described in the EMBOSS software package ( EMBOSS: European Open Package for Molecular Biology, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or higher) of the Needle program implementing the Needleman-Wunsch algorithm. The parameters used can be a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "longest identity" (obtained with the no-brief option) is used as percent identity and is calculated as follows:

(identical residues × 100)/(length of alignment - total number of gaps in alignment).

Optionally, the parameters used can be a gap opening penalty of 10, a gap extension penalty of 0.5 and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output marked "longest identity" (obtained with the non-simple option) is used as percent identity and calculated as follows:

(identical deoxyribonucleotides × 100)/(length of alignment - total number of gaps in alignment).

Expression: The term "expression" includes any step involved in producing a variant (polypeptide), including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that includes a polynucleotide encoding a variant (polypeptide) and is operably linked to regulatory sequences that provide its expression.

Fragment: The term "fragment" refers to a polypeptide that lacks one or more (eg, several) amino acids at the amino and/or carboxy terminus of a mature polypeptide; wherein the fragment still possesses the activities described herein (eg, alpha/beta Hydrolase activity, such as having the activity of hydrolyzing ZEN to HZEN).

The term "EC:3.1.1.-" as used herein means "zearalenone hydrolase" (also known as "zearalenone lactonase"), eg, capable of hydrolyzing ZEN to HZEN enzyme.

Host cell: The term "host cell" refers to any cell type that has a nucleic acid construct or expression vector comprising a polynucleotide of the invention, which is susceptible to transformation, transfection, transduction, and the like. The term "host cell" includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

Nucleic acid construct: The term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that is either isolated from a naturally occurring gene or modified to contain a nucleic acid fragment in a manner not found in nature or synthetically, It includes one or more regulatory sequences.

Operably linked: The term "operably linked" refers to a configuration in which the regulatory sequences are located at appropriate positions relative to the coding sequence of a polynucleotide, such that the regulatory sequences direct the performance of the coding sequence.

Control sequences: The term "control sequences" as used herein refers to nucleic acid sequences necessary for the performance of a polynucleotide encoding a variant (polynucleotide) of the invention. Each regulatory sequence may be native (ie, from the same gene) or foreign (ie, from a different gene) to the polynucleotide encoding the variant, or, native or exogenous to each other. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the regulatory sequences include a promoter and transcriptional and translational stop signals. To introduce specific restriction sites that facilitate ligation of the regulatory sequences to the coding regions of the polynucleotides of the invention, linkers are provided to the regulatory sequences.

As used herein, the term "corresponds to" refers to a method of determination of a sequence-specific amino acid in which a specific amino acid sequence is referenced (eg, US2020071638). For example, in the context of the present invention, when referring to a particular amino acid position, one skilled in the art can align another amino acid sequence with the referenced amino acid sequence in order to determine which particular amino acid is in of interest in the other amino acid sequence. The identification of corresponding amino acid residues in another alpha/beta hydrolase can be determined by alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison based on logarithmic expectations; 3.5 or higher; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (6.857 or higher; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899 -1900) and EMBOSS EMMA using ClustalW (version 1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective penalty parameters.

For purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid residues in another alpha/beta hydrolase. The amino acid sequence of another alpha/beta hydrolase was aligned with the mature polypeptide disclosed in SEQ ID NO: 1, based on the alignment, using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 1, the aforementioned Needleman-Wunsch algorithm using the EMBOSS software package (preferably 5.0.0 or higher) version) of the Needle program (EMBOSS: European Open Package for Molecular Biology, Rice et al., 2000, Trends Genet. 16: 276-277). The parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

The term "position" as used in accordance with the present invention refers to the position of an amino acid in the amino acid sequence described herein. As used herein, the term "corresponding" may include a position not only dependent on the numbering of the preceding nucleotide/amino acid.

As used herein, a "silent" mutation means a substitution of a base in a nucleic acid sequence that does not alter the amino acid sequence encoded by the nucleic acid sequence. "Conservative or equivalent" substitutions (or mutations) refer to substitutions as listed in "typical substitutions" in Table 1 below. "Highly conservative" substitutions as used herein are shown in Table 1 below under the heading "Preferred Substitutions".

Table 1. Amino Acid Replacement original Typical replacement better replacement Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu Leu (L) norleucine; ile; val; met; ala; ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; leu

Variant: The term "variant" refers to a polypeptide having a specific activity as described herein that includes alterations, ie substitutions, insertions and/or deletions, at one or more (eg, several) positions. Substitution means that the amino acid occupying a position is replaced by a different amino acid; deletion means that the amino acid occupying a position is removed; insertion means that the amino acid occupying a position is followed by another amino acid. amino acid.

In describing variants of the invention, the following nomenclature has been adjusted for ease of reference. The accepted one-letter or three-letter amino acid abbreviations for lUPAC are used.

replacement. For amino acid replacement, the following nomenclature is used: original amino acid, position, replaced amino acid. Therefore, substitution of Asn (N) at position 167 by Thr (T) is denoted as "N167T" or "Asn167Thr". Multiple mutations can be separated using additional markers ("+") or (","), e.g., "N167T+F168Y+S174C+F218Y" or "N167T, F168Y, S174C, F218Y", indicating multiple mutations at a given position replacement. In the examples of the present application, multiple mutations can be separated by commas, eg, N167T, F168Y, S174C, F218Y. Furthermore, "X" or "Xaa" as used herein can mean any amino acid (eg, as described in Table 1 above). Thus, "X167T" as used herein means that any amino acid at position 167 is replaced by T (Thr). Abbreviations may also be used to indicate only the position and substituted amino acid, provided that the original amino acid residue can be any amino acid residue. Thus, "X" or "Xaa" may be omitted when a substitution is indicated, eg, the "167T" designation may be used to indicate that any amino acid at position 167 is replaced by T (Thr). In addition, "X167G,A,S,C,U,I,L,V,T" as used herein means that any amino acid at position 167 is replaced by G, A, S, C, U, I, L, V or any permutation of T. Abbreviations may also be used to indicate only the original amino acid and its position, eg "N167", provided that the displacing amino acid residue can be any amino acid residue.

As used herein, the term "transgenic" refers to an organism whose genome has been altered by introducing exogenous genetic material or additional copies of autologous genetic material (eg, by transformation or recombination (eg, US7410800B2)). Transgenic organisms can be plants, mammals, fungi, bacteria or viruses. As used herein, "transgenic plant, seed or pollen grain" refers to a plant, seed or pollen grain, or any next generation progeny plant, seed or pollen grain derived therefrom, wherein the plant, seed or pollen grain or its The progeny contain introduced exogenous DNA that was not originally present in non-transgenic plants, seeds or pollen grains of the same variety. In addition, transgenic plants, seeds or pollen grains may contain sequences that were originally in the engineered plant, but in which the exogenous DNA has been altered so as to alter the level or pattern of expression of the coding sequence.

The term "recovering a variant" refers to purification of the variant from bacterial lysates, eg, by affinity purification.

The term "foodstuffs" refers to substances of edible value.

The term "fodder" refers to material fed to livestock.

The term "feed" refers to a substance used as food for livestock.

The term "additive" refers to a compound or substance that is added to other products or substances, eg, in small amounts, in order to affect desired properties and/or characteristics.

The term "prebiotic" refers to a compound or substance capable of inducing the growth and/or activity of beneficial microorganisms.

The term "antidote" refers to a compound or substance capable of reducing and/or inhibiting toxicity.

The term "nutritional supplement" refers to compounds or substances that supplement the nutritional content of a diet, eg, vitamins and minerals.

The term "intermediate" refers to a compound or substance produced in the process of obtaining the final product of the present invention (eg, at an intermediate stage of the process), eg, the food, forage, feed, additive (eg, food additive) of the present invention , forage or feed additives), antidotes, nutritional supplements or prebiotics.

The term "phycophytic substance" refers to substances derived from algal species.

Amino acid motif: The term "amino acid motif" or "motif" as used herein refers to a specifically defined stretch of amino acids of a polypeptide. Thus, an amino acid motif of the present invention refers to a short sequence of amino acids in a given polypeptide.

The term "OTA derivative" as used herein refers to a compound or substance having the following chemical structure:

In the molecular structure of the OTA derivative, R1, R2 and R3 can be any atom or group of atoms. In particular, R1 is selected from the group consisting of H and OH, R2 is selected from the group consisting of H and CH2-CH3, and R3 is selected from the group consisting of H and Cl. The OTA derivative can be: ochratoxin B, wherein R1 is H, R2 is H and R3 is H; ochratoxin C, wherein R1 is H, R2 is CH2-CH3 and R3 is Cl; or, ochratoxin TA, R1 is OH, R2 is H and R3 is Cl. In particular, the term "OTA derivative" refers to a compound or substance selected from the group consisting of Ochratoxin B and Ochratoxin C.

It must be noted that, as used herein, the singular forms "a" (a), "an" (an) and "the" (the) include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes reference to one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, capable of The methods described are modified or replaced.

Unless stated otherwise, the term "at least" preceding a series of elements should be understood to refer to each element of the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the embodiments of the invention described herein. Such equivalents are also intended to be included in the present invention.

Wherever used herein, the term "and/or" includes "and", "or" and "all or any other combination of the elements associated with the term."

The term "about" or "approximately" as used herein means within 20%, preferably within 10% and more preferably within 5% of a given value or range.

Throughout this specification and the scope of the appended claims, unless the context requires otherwise, the word "comprise" and variations thereof such as "comprises" and "comprising" will be understood to mean It is meant to include the specified integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps. As used herein, the term "comprising" may be replaced by the term "containing" or "including" or, as sometimes used herein, by the term "having" )replace.

As used herein, "consisting of" excludes any element, step, or ingredient not specified in the elements of the claim. As used herein, "consisting essentially of" does not exclude material or steps that do not materially affect the basic and novel characteristics of the claimed item.

In each instance herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by either of the other two terms.

The wild-type parental alpha/beta hydrolase of SEQ ID NO: 1 is capable of degrading ZEN to nontoxic HZEN, which exhibits a low melting point (Tm = 50°C) and has a dimeric homodimeric quaternary structure. Since high enzyme stability is a key point in the production and application of industrial enzyme products, there is a need for further development of improved α/β hydrolase variants that are capable of detoxifying ZEN at higher temperatures and are particularly suitable for use as food or feed additives or food or feed products.

In the process of the present invention, in the parental alpha/beta hydrolase having SEQ ID NO: 1, which directs the formation of a quaternary structure from homodimers to homotetramers and thus in particular enhances temperature stability Specific amino acid substitutions were identified. Accordingly, the present invention relates to the use of the improved hydrolase variants described herein for degrading zearalenone (ZEN).

ZEN (CAS Reg. #17924-92-4, (4S,12E)-16,18-dihydroxy-4-methyl-3-oxabicyclo[12.4.0]octadeca-1(14),12 ,15,17-tetraene-2,8-dione) is a non-steroidal estrogenic macrolide with the following structural formula, synthesized through the polyketone metabolic pathway:

However, various ZEN derivatives also occur in nature and can be formed by enzymatic or chemical modification of ZEN. Examples include glycoside ZEN conjugates, or those containing sulfate, formed from ZEN metabolites that are metabolized by fungi, plants, or mammals, as well as in the human or animal body, and the like. ZEN derivatives are hereinafter understood to be ZEN conjugates or ZEN metabolites that are naturally occurring or synthesized by chemical or biochemical synthesis, in particular α-zearalenol (α-ZEL; CAS Registry No. 36455 -72-8;(2E,7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]-octadeca-1(18),2,14 , 16-tetraen-13-one), β-zearalenol (β-ZEL; CAS Reg. #71030-11-0; (2E,7S,11S)-7,15,17-trihydroxy- 11-Methyl-12-oxabicyclo[12.4.0]octadec-1(18),2,14,16-tetraen-13-one), α-zearatol (α-ZAL; CAS Registration number # 26538-44-3; (7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14, 16-trien-13-one), β-zearatol (β-ZAL; CAS Reg. # 42422-68-4; (7S,11S)-7,15,17-trihydroxy-11-methyl) -12-oxabicyclo[12.4.0]-octadec-1(14),15,17-trien-13-one), zearalenone 14-sulfate (Z14S; [(2E,11S )-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadec-1(18),2,14,16-tetraene-17-yl] bisulfate), zearalenone-14-glycoside (Z14G; (2E,11S)-15-hydroxy-11-methyl-17-[(3R,4S,5S,6R)-3,4,5 -Trihydroxy-6-(hydroxymethyl)-tetrahydropyran-2-yl]oxy-12-oxabicyclo[12.4.0]octadeca 1(18)2,14,16-tetraene-7 , 13-dione), and zearalenone (ZAN; CAS Reg. # 5975-78-0; (11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo-[12.4. 0] octadeca-1(18),14,16-triene-7,13-dione).

ZEN and ZEN derivatives especially α-ZEL, β-ZEL, Z14S, α-ZAL, β-ZAL, Z14G and ZAN can also be detected in processed food or animal feed products, such as bread or beer, because of their High chemical and physical stability.

Any polypeptide with sequence numbers 1 to 35 can successfully hydrolyze ZEN or ZEN derivatives. It is believed that the hydrolysis of ZEN or its derivatives occurs at the ester group according to the following reaction mechanism:

Hydrolysis of ZEN to form non-toxic hydrolyzed zearalenone (HZEN) and/or hydrolyzed ZEN derivatives can occur using the alpha/beta-hydrolases of the present invention. Further decarboxylation of HZEN to give decarboxylated hydrolyzed ZEN (DHZEN) and/or decarboxylated hydrolyzed ZEN derivatives is believed to be spontaneous.

The alpha/beta-hydrolases and variants described herein are capable and suitable for degrading ZEN. For example, alpha/beta-hydrolases can be adapted for cofactor-free hydrolytic cleavage of ester groups of ZEN and/or derivatives thereof (eg, hydrolysis is _H2O dependent).

ZEN degradation can be measured as follows: Zearalenone degradation assay buffer (118.5 mM NaCl, 8.55 mM acetic acid, 14.9 mM acetate, 0.1 mg/ml BSA, pH 5.0, (Jantratid, E., Janssen, N., Reppas, C. & Dressman, J. B. (2008): Dissolution media simulating conditions in the proximal human gastrointestinal tract: An update. Pharmaceutical Research 25(7): 1663-1576), with 0.1 mg/ml bovine serum albumin and 0.52 ppm zearalenone as substrate), pre-warmed at 37.0 °C, and transfer 960 µl of assay buffer to a reaction tube in a 96-deep-well plate. The plate was sealed with a removable lid and stored in a DWP-thermal shaker at 37.0°C until the start of the zearalenone degradation assay. Use purified zearalenone-cleavage polypeptides by diluting in sample buffer (Teorell Stenhagen buffer pH 7.5, containing 0.1 mg/ml bovine serum albumin) to concentrations higher than those analyzed in the final zearalenone degradation experiments 25 times higher to prepare the enzyme working solution, the concentration of zearalenone-cleaving polypeptide can effectively degrade zearalenone under the given conditions. To begin the zearalenone degradation assay, add 40.0 µl of the enzyme working solution to a tube containing 960.0 µl of assay buffer to achieve a final ZEN concentration of 0.5 ppm in the assay reaction. The addition of pH 7.5 enzyme working solution did not alter the pH 5.0 zearalenone degradation reaction. The zearalenone degradation reaction was incubated in a DWP-thermal shaker with continuous shaking at 37.0°C. Immediately after the zearalenone degradation reaction started, resuspend and mix with a pipette tip, transfer 120.0 µl of the 0.0 h sample to a tube on a new PCR plate, and seal it with a tube cap. Additional samples were taken from the zearalenone degradation reaction after several time points (eg, 5.0, 10.0, 20.0, 30.0, 45.0, 60.0, 90.0 min). Once the sample is removed from the degradation reaction, thermally inactivate zearalenone cleavage in the sample by incubating it in a thermo-block (eg, Thermal Shake lite, 460-029, VWR) for 10.0 minutes at 99.0°C peptide. Subsequently, the tubes were centrifuged (3.0 min, room temperature, 2500 x g) and 90.0 µl of the supernatant was transferred to a rimmed PCR plate (e.g., twin.tec® PCR plate, 0030128648, Eppendorf AG), which was sealed with a heat-sealed film (for example, heat-sealing film, 0030127838, Eppendof AG) to seal. These sample plates were stored at 4.0°C until HPLC-FLD measurement. Zearalenone concentrations were measured according to a modified HPLC-FLD method described by Vekiru et al. (Vekiru et al. (2016) 'Isolation and characterization of enzymatic zearalenone hydrolysis reaction products' World Mycotoxin Journal 9:353-363). Extinction at 278.0 nm and emission at 465.0 nm, analyzed on HPLC-FLD. The retention time for zearalenone was 1.8 minutes when separated on a Kinetex 5µm EVO C18 100 A 50.0 x 2.1 mm column (00B-4633-AN, Phenomenex Inc.) at 40.0°C using the following method : Using solvent A (95.0 % acetonitrile + 4.9 % water + 0.1 % formic acid) and solvent B (5.0 % acetonitrile + 94.9 % water + 0.1 % formic acid), gradient: 0.0-0.5 min 10 % phase B, 0.5-2.0 min linear Increase to 50.0 % Phase B, then reduce to 10 % Phase B in 0.1 min, continuing for a total run time of 2.3 min. The flow rate was set to 1.5 ml/min and the injection volume was 5.0 μl. Similar acquisition settings were used for the various analyses. Zearalenone was quantified based on the calibration of the zearalenone external standard. The zearalenone concentration (µM) of the reaction samples taken is plotted against the sampling time point. The slope of linear zearalenone degradation over time was calculated as μmol zearalenone decreased per minute in the volume of the enzymatic reaction. Taking into account possible dilutions of purified zearalenone-cleavage polypeptides and including these appropriate dilution factors in the calculation, calculate in µmol zearalenone degraded per minute per mg of purified enzyme (µmol ZEN/min). /mg) expressed the specific enzymatic activity (specific enzymatic activity).

In this context, it should be noted that the term "unit" or "U" refers to the measurement of the catalytic activity of an enzyme, defined as the reaction or cleavage of substrate per minute under set conditions (in the present invention, zein red Micromoles (µmol) of mycolene). The enzyme concentration of the enzyme or polypeptide solution is defined by the "activity" of the enzyme or polypeptide solution, expressed in units per milliliter (U/ml) or units per liter (U/l) of the solution.

In some embodiments, the invention provides variants of the parental alpha/beta hydrolase comprising substitutions at one or more positions corresponding to SEQ ID NO: 1 (preferably using SEQ ID NO: 1 number) of 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59 , 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304, wherein the variant has alpha/beta hydrolase activity, and wherein the variant is With the polypeptide of SEQ ID NO: 1 has at least 71% (preferably: at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% %, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%) but Polypeptides of less than 100% sequence identity, preferably, the variant has hydrolase EC: 3.1.1.-activity.

In other embodiments, the variants of the present invention have one or more of the following properties: (i) improved properties relative to the parent alpha/beta hydrolase, wherein the improved properties include increased temperature stability, preferably ground, compared to SEQ ID NO: 1, improved temperature stability; (ii) capable of tetramerization, preferably, having a tetramer quaternary structure, further preferably, the tetramerization is homotetramerizes; (iii) has a melting point (Tm) greater than 50°C, preferably the Tm ranges from about 51°C to about 70°C; (iv) is capable of degrading zearalenone and/or Derivatives thereof; (v) having at least one (preferably at least two, more preferably at least three, most preferably at least four) amino acid motifs associated with an amine selected from The amino acid sequence has at least 80% (preferably at least 83%, more preferably at least 90%) sequence identity: (a) SEQ ID NO: 36 (QVDLGEX ₁ X ₂ MN), wherein X ₁ is V or I, preferably V, and wherein X ₂ is V or T, preferably V; (b) SEQ ID NO: 37 (EYDPEWX ₃ RX ₄ FX ₅ EGTV) wherein X ₃ is G or A, preferably A, and wherein X ₄ is A or V, preferably A, and wherein X ₅ is F or Y; (c) SEQ ID NO: 38 (MLX ₆ QVKTPX ₇ LX ₈ THH), wherein X ₆ is S or G, preferably S, and wherein X is I or V, preferably I, and wherein X is I or L, preferably I; and (d) SEQ ID NO: 39 (PAX _{9 LLX 10 PEQTGSWWSYEX 11} X ₁₂ X ₁₃ GLLX ₁₄ EX ₁₅ FHVX ₁₆ AVDX ₁₇ RGQGRSX ₁₈ WTPX ₁₉ RYSLDNFGNDLVRFIX ₂₀ LVX ₂₁ KRPVX ₂₂ VX ₂₃ GNSSGGX ₂₄ LAAWLSAYX ₂₅ MPGQX ₂₆ RX ₂₇ X ₂₈ LCEDX ₂₉ X ₃₀ FFASELVPAX ₃₁ GHSVX ₃₂ QX ₃₃ AGPX ₃₄ FELX ₃₅ R)，其中, X ₉ is V or L, preferably L, and wherein X ₁₀ is L or I, preferably L, and wherein X ₁₁ is P or E, preferably P, and wherein X ₁₂ is V or A, Preferably V, and wherein _X is I or M, preferably I, and wherein _X is A or S, preferably A, and wherein _X is S, N or H, preferably S, and wherein , X ₁₆ is F or Y, preferably F, and wherein X ₁₇ is I or L, preferably I, and wherein X ₁₈ is T or S, Preferably T, and wherein X is K or R, preferably K, and wherein _X is S, _A or N, preferably S, and wherein _X is V or I, preferably V, and wherein , X ₂₂ is I or V, preferably I, and wherein X ₂₃ is S or A, preferably S, and wherein X ₂₄ is V or L, preferably V, and wherein X ₂₅ is A or S, Preferably A, and wherein _X is I or L, preferably I, and wherein _X is A or G, preferably A, and wherein _X is A or V, preferably A, and wherein X ₂₉ is T, A or P, preferably T, and wherein X ₃₀ is P or A, preferably P, and wherein _X is Y or H, preferably H, and wherein _X is L or R, R is preferred, and wherein X ₃₃ is A or G, preferably A, and wherein X ₃₄ is A or V, preferably A, and wherein X ₃₅ is Y or F, preferably Y.

In other embodiments, variants of the invention include one or more of the following substitutions, or equivalent (eg, conservative) amino acid substitutions thereof (eg, as described in Table 1 herein): X167T, X168Y, X174C, X218Y; X167T; X167S; X167A; X167C; X167L; X167T, X174T; X167T, X174C; X155K, X167T, X174C, X175A; X155K, X167T, X174C, X175T; X167S, X174C; X167T, X174C, X218Y; X167T, X168Y, X174C; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X268L, X306I; X10N, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X17Y, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X28T, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X39R, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X47S, X50E, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X57E, X58A, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X63S, X65H, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X69Y, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X126S, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X139P X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y X230R; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X245V; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X251E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X281E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X287T, X289S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X293S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X304R; X10N, X17Y, X28T, X39R, X57E, X58A, X59M, X63S, X65H, X69Y, X126S, X139P, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225, X28, X230 X289S, X293S, X304R; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X202V, X216V, X218Y, X287T, X289S; X155R, X167T, X168Y, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V; X155R, X167T, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y; X155R, X167T, X168Y, X174C, X175V, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X306I; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X287S, X289S, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X287S; or its equivalent (eg, conservative) amino acid substitutions (eg, those described in Table 1 herein).

In other embodiments, the parental alpha/beta hydrolase (preferably having EC: 3.1.1.-hydrolase activity) of the invention is selected from the group consisting of the following polypeptides: (i) the polypeptide with SEQ ID NO: 1 have at least 60% (preferably, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% , or at least 99.5%, or 100%) polypeptides of sequence identity; (ii) fragments of the polypeptides of SEQ ID NO: 1, wherein the fragments have alpha/beta hydrolase activity, preferably EC: 3.1.1.- Hydrolase activity.

In other embodiments, variant (i) of the present invention comprises a permutation selected from or selected from the group consisting of the following permutations: N167T, F168Y, S174C, F218Y; N167T; N167S; N167A; N167C; N167L; N167T, S174T; N167T, S174C; R155K, N167T, S174C, V175A; R155K, N167T, S174C, V175T; N167S, S174C; N167T, S174C, F218Y; N167T, F168Y, S174C; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, Q268L, V306I; D10N, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F17Y, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; V28T, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; H39R, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; E47S, G50E, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; P57E, V58A, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A63S, S65H, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F69Y, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A126S, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; T139P N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, L230R; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A245V; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H251E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A281E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, L287T, M289S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S293S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S304R; D10N, F17Y, V28T, H39R, P57E, V58A, I59M, A63S, S65H, F69Y, A126S, T139P, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225, L28ER, A28ER M289S, F293S, S304R; and/or (ii) is selected from the group consisting of SEQ ID NOs: 2-35.

In other embodiments, the present invention provides for obtaining variants of the parental alpha/beta hydrolase (preferably having EC: 3.1.1.-hydrolase activity) and/or increasing said parental alpha/beta hydrolase A method of stability, the method comprising: at positions 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28 with SEQ ID NO: 1 , 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304 (preferably using SEQ ID NO: 1 numbering), introducing substitutions into the parental alpha/beta hydrolase at one or more corresponding positions, wherein the variant has alpha/beta hydrolase activity; and, recovering the variant; preferably, the parental alpha The /β hydrolase is according to any one of the preceding claims, and further preferably, the parental α/β hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1.

In other embodiments, the present invention relates to polynucleotides encoding variants of the present invention. In other embodiments, the present invention relates to nucleic acid constructs comprising the polynucleotides of the present invention. In other embodiments, the present invention relates to nucleic acid constructs comprising the polynucleotides of the present invention. In other embodiments, the present invention relates to recombinant host cells comprising at least one of: (i) a variant and/or parental alpha/beta hydrolase of the present invention; (ii) a polynucleotide of the present invention; ( iii) a nucleic acid construct of the invention; and/or, (iv) an expression vector of the invention.

Accordingly, the present invention also relates to nucleic acid molecules encoding the alpha/beta hydrolase variants described herein. Nucleic acids can be introduced or inserted into expression vectors. The term "expression vector" refers to a nucleic acid molecular construct capable of expressing a gene in vivo or in vitro. In particular, it may include a DNA construct suitable for transferring a polypeptide-encoding nucleotide sequence into a host cell (eg, a recombinant host cell) for integration into the genome or episomal extrachromosomal space, and for intracellular expression of the polypeptide- The encoding nucleotide sequence, and optionally, the polypeptide is transported extracellularly. Expression vectors as described herein can be expressed in host cells. The term "host cell" refers to all cells containing the nucleotide sequence or expression vector to be expressed, which are capable of producing the enzymes or polypeptides according to the invention. In particular, this refers to prokaryotic cells and/or eukaryotic cells, preferably Pichia, Escherichia coli, Bacillus subtilis, Streptomyces, Hansenula, Trichoderma, Lactobacillus, Aspergillus, plant cells and/or Spores of Bacillus, Trichoderma or Aspergillus. The name P. pastoris used herein is synonymous with the name Komagataella pastoris , P. pastoris being the older name and K. pastoris being the newer system Title (Yamada et al. (1995) 'The Phylogenetic Relationships of Methanol-assimilating Yeasts Based on the Partial Sequences of 18S and 26S Ribosomal RNAs: The Proposal of Komagataella Gen. Nov. (Saccharomycetaceae)' Bioscience, Biotechnology and Biochemistry, Vol. 59, issue 3, pp. 439-444). In particular, the species of Komagataella pastoris has recently been redefined as Komagataella phaffii (Kurtzman (2009) “Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis). "J Ind Microbiol Biotechnol. 36(11):1435-8). As used herein, Komagataella phaffii refers , for example, to the strains Komagataella phaffii CBS 7435, Komagataella phaffii GS115 or Komagataella phaffii JC308.

In other embodiments, the present invention relates to transgenic plants, transgenic seeds or transgenic pollen grains comprising one or more (preferably more) of the following: (i) variants of the present invention and/or parental alpha/beta hydrolysis (ii) polynucleotides of the invention; (iii) nucleic acid constructs of the invention; (iv) expression vectors of the invention; and/or (v) recombinant host cells of the invention.

In other embodiments, the present invention relates to food, intermediate food; forage, intermediate forage; feed, intermediate forage; additive (preferably, food additive, forage additive or feed additive), intermediate additive (preferably, food intermediate additive , forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculation bacteria; antidote; veterinary composition; pharmaceutical composition , and/or mixtures thereof, which include one or more of the following: (i) a variant of the present invention and/or a parent alpha/beta hydrolase; preferably, the parent alpha/beta hydrolase and SEQ ID The polypeptide of NO: 1 has at least 95% sequence identity; (ii) the polynucleotide of the present invention; (iii) the nucleic acid construct of the present invention; (iv) the expression vector of the present invention; (v) the recombinant of the present invention host cells; (vi) transgenic plants, transgenic seeds and/or transgenic pollen grains of the invention.

In other embodiments, the present invention relates to a composition or kit comprising one or more of: (i) a variant of the present invention and/or a parent alpha/beta hydrolase; preferably, the parent The alpha/beta hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1; (ii) the polynucleotide of the present invention; (iii) the nucleic acid construct of the present invention; (iv) the expression vector of the present invention (v) recombinant host cells of the present invention; (vi) transgenic plants, transgenic seeds and/or transgenic pollen grains of the present invention; and/or (vii) foods, intermediate foods; forages, intermediate forages; feed, Intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, Intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; pharmaceutical compositions, and/or mixtures thereof.

Accordingly, the present invention also relates to compositions comprising the alpha/beta hydrolase variants described herein. Preferably, the composition may be a food or feed additive or food or feed product. Methods of preparing these food and/or feed compositions are known to those skilled in the art and are described in particular in WO 99/35240.

In other embodiments, the compositions or kits of the present invention further comprise a nutritionally acceptable carrier (eg, water) and/or a parental alpha/beta hydrolase of the present invention, preferably, the parental alpha /β hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1.

In some other embodiments, the compositions or kits of the invention further comprise one or more of the following: (i) capable of removing OTA and/or at least one OTA derivative (eg, Ochratoxin B and/or Ochratoxin C) ) toxicity of one or more other polypeptides, further preferably, the one or more other polypeptides belong to the M20 peptidase aminoamylase 1-like protein 2-like aminohydrolase subfamily (for example, M20 peptidase ACY1L2 amino The hydrolase subfamily, for example, according to the conserved domain database (eg https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd05672) identifier is cd05672, and/or, has aminopeptidase activity (eg EC 3.4.11.10), and/or, including carboxypeptidase activity (eg carboxypeptidase A and/or B activity, eg with EC 3.4.17.1 and/or EC 3.4.17.2, respectively), and /or, thermolysin activity (eg with EC 3.4.24.27); (ii) capable of removing one or more mycotoxins (eg, ZEN and/or trichothecenes such as deoxynivalenol) ), nivalenol, neosolaniol, trichotecin, crotocin, roridin A, staphylocin H (satratoxin H), diacetoxyscirpenol, HT-2 toxin or T-2 toxin; aflatoxins such as aflatoxins B1, B2, G1 or G2; fumonisins such as Fumonisins B1, B2, B3 or B4; polypeptide mycotoxins such as beauvericin or enfusaricin; zearalenone; citrin; patulin; ergot alkaloids such as ergotamine) and/or one or more other polypeptides for the toxicity of one or more plant- and/or bacterial-derived toxins (eg endotoxins, etc.), in particular, the one or more other polypeptides capable of removing one or more other mycotoxins and/or or one or more plant- and/or bacterial-derived toxins, eg, fumonisin esterase (eg, as disclosed in WO 2016/134387 A1) and/or zearalenone lactonase (eg, as disclosed in WO 2016/134387 A1) WO 2020/025580 A1) and/or ergopeptidase (e.g. as disclosed in WO 2014/056006 A1); (iii) one or more organic adsorbents (e.g. live, inactivated, Lyophilized, dormant and/or dead whole yeast, or yeast-derived products such as yeast cell walls, or yeast oligomers such as mannan) and/or one or more inorganic adsorbents (eg, diatomaceous earth and/or Clay minerals such as kaolin or kaolinite , montmorillonite such as montmorillonite, illite or chlorite; in particular, bentonite); (iv) capable of removing one or more other mycotoxins (eg, trichothecene mycotoxins such as deoxynivalenol, citrullus Physalisol, Neosolanisol, Mucormycin, Flatworm Toxin, Bacillus A, Staphylococcus H, Diacetoxyfenol, HT-2 Toxin or T-2 Toxin ; aflatoxins, such as aflatoxins B1, B2, G1 or G2; fumonisins, such as fumonisins B1, B2, B3 or B4; polypeptide mycotoxins, such as beauverin or enfusarium ; zearalenone; citrinin; patulin; ergot alkaloids, such as ergotamine) and/or one of the toxicity of one or more plant- and/or bacterial-derived toxins (eg, endotoxins, etc.) or a plurality of live, inactivated, lyophilized and/or dormant microorganisms, in particular selected from the group consisting of Trichosporon and Apiotrichum (eg as disclosed in WO 03/053161 A1) and Rhododendron (for example, as disclosed in EP 3 501 526 A1); (v) one or more plant products (for example, seaweed, preferably seaweed meal); and/or, algae, preferably seaweed meal (algae meal); and/or, thistle, preferably thistle seed; and/or, licorice preparation, preferably licorice powder and/or licorice extract, for example, as disclosed in WO 2018/121881 A1); (vi) a or more flavoring compounds (e.g., plant extracts such as from oregano, thyme, wintergreen, coriander, marjoram, peppermint, peppermint, fennel, citrus, lemon, cumin, star anise, cloves, cinnamon, and/or garlic and/or, essential oils such as D-limonene, gamma-terpenes, p-cymene, 2-carene, linalool oxide, isomenthone, camphor, linalool, Terpinen-4-ol, 2-isopropyl-1-methoxy-4-toluene, L-menthol, ethylamine, alpha-terpineol, beta-caryophyllene, D-carvone, water (vii) one or more vitamins (e.g., vitamins A, D, E, K, C, B1, B2, B3, B4, B5, B6, B7, B8, B9 and/or B12; especially vitamin E).

In particular embodiments, the compositions or kits of the present invention further comprise one or more of the following: bentonite, fumonisin esterase and/or zearalenone lactonase, capable of removing one or more mycotoxins Toxic micro-organisms of the Coriobacteriaceae family (for example, microorganisms selected from the Coriobacteriaceae family, e.g. https://lpsn.dsmz.de/family/coriobacteriaceae), diatomaceous earth, yeast (especially inactivated yeast), seaweed flour , thistle seeds, and one or more flavoring compounds.

In some other embodiments, a composition (eg, typical compositions 1-24 set forth in Table 2 below) or kits (corresponding to typical compositions 1-24 set forth in Table 2 below) of the invention comprise one or Various other components (eg, in addition to at least one polypeptide according to the invention). Such other exemplary compositions or kits are specifically disclosed in Table 2 below, which describes embodiments of the present invention.

Table 2. Typical compositions or kits of the invention including one or more additional components. composition number 1 2 3 4 5 6 Any variant or polypeptide according to the invention (eg, SEQ ID NOs: 2-35) Yes Yes Yes Yes Yes Yes Any suitable polypeptide capable of detoxifying one or more other mould-/plant-/bacterial-toxins For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof any suitable organic adsorbent For example, inactivated yeast For example, dry yeast For example, yeast any suitable inorganic adsorbent For example, bentonite For example, zeolite For example, bentonite-montmorillonite For example, bentonite For example, bentonite For example, bentonite Any suitable microorganism capable of detoxifying one or more other mycotoxins For example, Corio-bacteriaceae For example, Rhododendron For example, Rhododendron any suitable plant product For example, seaweed powder For example, thistle seed any suitable vitamin For example, vitamin E any suitable flavoring compound Yes composition number 7 8 9 10 11 12 Any variant or polypeptide according to the invention (eg, SEQ ID NOs: 2-35) Yes Yes Yes Yes Yes Yes Any suitable polypeptide capable of detoxifying one or more other mould-/plant-/bacterial-toxins For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof For example, fumonisin esterase, eg, having EC: 3.1.1.87 enzymatic activity, eg, UniProtKB-D2D3B6, and/or, variants thereof any suitable organic adsorbent For example, inactivated yeast For example, Saccharomyces cerevisiae any suitable inorganic adsorbent For example, bentonite For example, bentonite For example, bentonite For example, bentonite For example, bentonite For example, bentonite Any suitable microorganism capable of detoxifying one or more other mycotoxins For example, Eubacterium sp. For example, Rhododendron For example, Eubacterium For example, Rhododendron any suitable plant product For example, seaweed extract For example, seaweed powder, milk thistle For example, algal matter any suitable vitamin any suitable flavoring compound composition number 13 14 15 16 17 18 Any variant or polypeptide according to the invention (eg, SEQ ID NOs: 2-35) Yes Yes Yes Yes Yes Yes Any suitable polypeptide capable of detoxifying one or more other mould-/plant-/bacterial-toxins For example, fumonisin esterase, for example, having EC: 3.1.1.87 enzymatic activity, for example, UniProtKB-D2D3B6, and/or, a variant thereof, zearalenone lactonase, for example, UniProtKB-Q8NKB0 and/or , its variant For example, fumonisin esterase, for example, having EC: 3.1.1.87 enzymatic activity, for example, UniProtKB-D2D3B6 and/or, a variant thereof, zearalenone lactonase, for example, UniProtKB-Q8NKB0 and/or, its variants any suitable organic adsorbent For example, inactivated yeast For example, dry yeast For example, yeast any suitable inorganic adsorbent For example, bentonite For example, zeolite For example, bentonite-montmorillonite For example, bentonite For example, bentonite For example, bentonite Any suitable microorganism capable of detoxifying one or more other mycotoxins For example, Rhododendron For example, Rhododendron For example, Rhododendron any suitable plant product For example, seaweed powder any suitable vitamin For example, vitamin E any suitable flavoring compound Yes composition number 19 20 twenty one twenty two twenty three twenty four Any variant or polypeptide according to the invention (eg, SEQ ID NOs: 2-35) Yes Yes Yes Yes Yes Yes Any suitable polypeptide capable of detoxifying one or more other mould-/plant-/bacterial-toxins Fumonisin esterase, e.g., having EC: 3.1.1.87 enzymatic activity, e.g., UniProtKB-D2D3B6, and/or, a variant thereof, zearalenone lactonase, e.g., UniProtKB-Q8NKB0, and/or, its variants Fumonisin esterase, e.g., having EC: 3.1.1.87 enzymatic activity, e.g., UniProtKB-D2D3B6, and/or, a variant thereof, zearalenone lactonase, e.g., UniProtKB-Q8NKB0, and/or, its variants Fumonisin esterase, e.g., having EC: 3.1.1.87 enzymatic activity, e.g., UniProtKB-D2D3B6, and/or, a variant thereof, zearalenone lactonase, e.g., UniProtKB-Q8NKB0, and/or, its variants Fumonisin esterase, e.g., having EC: 3.1.1.87 enzymatic activity, e.g., UniProtKB-D2D3B6, and/or, a variant thereof, zearalenone lactonase, e.g., UniProtKB-Q8NKB0, and/or, its variants any suitable organic adsorbent For example, inactivated yeast For example, Saccharomyces cerevisiae any suitable inorganic adsorbent For example, bentonite For example, bentonite For example, bentonite For example, bentonite For example, bentonite For example, bentonite Any suitable vitamin capable of detoxifying one or more other mycotoxins For example, Eubacterium For example, Rhododendron For example, Eubacterium For example, Rhododendron any suitable plant product For example, seaweed extract For example, seaweed powder, milk thistle For example, algal matter any suitable vitamin any suitable flavoring compound

In other embodiments, the variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, recombinant host cell, transgenic plant, transgenic seed, transgenic pollen grain, food product, intermediate food product of the invention; Forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate Antidote; nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, silage inoculum; antidote; veterinary composition; pharmaceutical composition and/or mixtures thereof, compositions and/or kits, Suitable for use as a medicine and/or in treatment, preferably, in the prevention or treatment of diseases, further preferably, the parent α/β hydrolase and the polypeptide of SEQ ID NO: 1 have at least 95 % sequence identity.

In other embodiments, the variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, recombinant host cell, transgenic plant, transgenic seed, transgenic pollen grain, food product, intermediate food product of the invention; Forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate Antidote; nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, silage inoculum; antidote; veterinary composition; pharmaceutical composition and/or mixtures thereof, compositions and/or kits, It is suitable for preventing and/or treating fungal poisoning, preferably, it is suitable for preventing and/or treating hormonal disorders caused by zearalenone or its derivatives.

In other embodiments, the present invention relates to variants, parental alpha/beta hydrolases, polynucleotides, nucleic acid constructs, expression vectors, recombinant host cells, transgenic plants, transgenic seeds, transgenic pollen grains, food products, Intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); detoxification Agents, Intermediate Antidotes; Nutritional Supplements, Intermediate Nutritional Supplements; Prebiotics, Intermediate Prebiotics, Silage Inoculants; Antidote; Veterinary Compositions; Pharmaceutical Compositions and/or mixtures thereof for use or as One or more of: (i) degradation of zearalenone and/or its derivatives; (ii) food, food, intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, Silage inoculum; antidote; veterinary composition; pharmaceutical composition; (iii) manufacture/production of one or more of the following: food, intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably , food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, Intermediate prebiotics, silage inoculants; antidote; veterinary compositions; pharmaceutical compositions and/or mixtures thereof; (iv) manufacture/production of silage, biogas, bioethanol or sugar, preferably from sugar cane or Sugar beet manufacturing/production; (v) prevention and/or treatment of mold poisoning.

In other embodiments, the present invention provides methods of degrading zearalenone and/or derivatives thereof, comprising: (a) providing one or more of: (i) a variant and/or parent of the present invention α/β hydrolase, preferably, the parent α/β hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1; (ii) the polynucleotide of the present invention; (iii) the present Nucleic acid constructs of the invention; (iv) expression vectors of the invention; (v) recombinant host cells of the invention; (vi) transgenic plants, transgenic seeds and/or transgenic pollen grains of the invention; (vii) food products of the invention , intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); Antidote, intermediate antidote; nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, silage inoculum; antidote; veterinary composition; pharmaceutical composition and/or mixtures thereof; and/or ( vii) a composition or kit of the invention; (b) contacting one or more substances from (a) with zearalenone and/or a derivative thereof (eg, to form an enzyme-substrate mixture).

It is to be understood that this invention is not limited to the particular methods, procedures, reagents, etc. described herein, which may vary. The technical terms used herein are only intended to describe specific embodiments, and are not intended to limit the protection scope of the present invention, which is only defined by the scope of the patent application.

Publications and patents (including all patents, patent applications, scientific publications, manufacturer's instructions, instructions for use, etc.) cited throughout this specification, whether above or below, are hereby incorporated by reference in their entirety . Nothing herein is to be construed as an admission that the present invention is not entitled to prior disclosure by virtue of prior invention. To the extent that material included by reference is inconsistent or inconsistent with this specification, this specification supersedes any such material.

The invention is also characterized by the following items: 1. A variant of a parental alpha/beta hydrolase comprising substitutions at one or more positions corresponding to: SEQ ID NO: 1 (preferably Using the numbering of SEQ ID NO: 1) 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58 , 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304, wherein the variant has alpha/beta hydrolase activity (e.g. zearalenone) hydrolase), and wherein the variant is at least 71% (preferably: at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%) with the polypeptide of SEQ ID NO: 1 %, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5 %, at least 99%, or at least 99.5%) but less than 100% sequence identity, preferably the variant has a hydrolase EC: 3.1.1.-activity. 2. The variant according to any preceding item, wherein the variant has one or more of the following properties: i) an improved property relative to the parent alpha/beta hydrolase, wherein the improved property comprises an increased temperature stability, preferably improved temperature stability compared to SEQ ID NO: 1; ii) the variant is capable of tetramerization, preferably has a tetrameric quaternary structure, further preferably, the The tetramerization is homotetramerization; iii) the melting point (Tm) is greater than 50°C, preferably, the Tm is in the range of about 51°C to about 70°C; iv) capable of degrading zearalenone and/or derivatives thereof; v) comprising at least one (preferably at least two, More preferably at least three, most preferably at least four) amino acid motifs: a) QVDLGEX ₁ X ₂ MN (SEQ ID NO: 36), wherein X ₁ is V or I, preferably V, and wherein X ₂ is V or T, preferably V; b) EYDPEWX ₃ RX ₄ FX ₅ EGTV (SEQ ID NO: 37), wherein X ₃ is G or A, preferably A, and wherein X ₄ is A or V, Preferably A, and wherein X ₅ is F or Y; c) MLX ₆ QVKTPX ₇ LX ₈ THH (SEQ ID NO: 38), wherein X ₆ is S or G, preferably S, and wherein X ₇ is I or V, preferably I, and wherein X ₈ is I or L, preferably I; d) PAX ₉ LLX ₁₀ PEQTGSWWSYEX ₁₁ X ₁₂ X ₁₃ GLLX ₁₄ EX ₁₅ FHVX ₁₆ AVDX ₁₇ RGQGRSX ₁₈ WTPX ₁₉ RYSLDNFGNDLVRFIX ₂₀ LVX ₂₁ KRPVX ₂₂ VX ₂₃ GNSSGGX ₂₄ LAAWLSAYX ₂₅ MPGQX ₂₆ RX ₂₇ X ₂₈ LCEDX ₂₉ X ₃₀ FFASELVPAX ₃₁ GHSVX ₃₂ QX ₃₃ AGPX ₃₄ FELX ₃₅ R (SEQ ID NO: 39) wherein X ₉ is V or L, preferably L, and wherein _X10 is L or I, preferably L, and wherein _X11 is P or E, preferably P, and wherein _X12 is V or A, preferably V, and wherein _X13 is I or M, preferably Preferably I, and wherein _X is A or S, preferably _A , and wherein _X is S, N or H, preferably S, and wherein X is F or Y, preferably F, and wherein X ₁₇ is I or L, preferably I, and wherein X ₁₈ is T or S, preferably T, and wherein X ₁₉ is K or R, preferably K, and and, wherein X ₂₀ is S, A or N, preferably S, and wherein X ₂₁ is V or I, preferably V, and wherein X ₂₂ is I or V, preferably I, and wherein X ₂₃ is S or _A , preferably S, and wherein X is V or L, preferably V, and wherein _X is A or S, preferably A, and wherein _X is I or L, preferably I, and, wherein X ₂₇ is A or G, preferably A, and wherein X ₂₈ is A or V, preferably A, and wherein X ₂₉ is T, A or P, preferably T, and wherein X ₃₀ is P or A, preferably P, and wherein _X is Y or H, preferably H, and wherein _X is L or R, preferably R, and wherein _X is A or G, preferably A, Also, wherein X ₃₄ is A or V, preferably A, and wherein X ₃₅ is Y or F, preferably Y. 3. The variant according to any of the foregoing, wherein the number of substitutions is 1-30, preferably 1-20 and 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 permutations. 4. The variant according to any one of the preceding paragraphs, wherein the variant comprises substitutions at one or more positions corresponding to the following positions of SEQ ID NO: 1 (for example, each row of the following table represents a feature according to the invention) A single typical combination of, for example, a variant of a particular embodiment): N167, F168, S174, F218; N167; N167, S174; R155, N167, S174, V175; N167, S174; N167, S174, F218; N167, F168, S174; N167, F168, S174, D179, A182, A183, K186, A187, F218; N167, F168, S174, D179, A182, A183, K186, A187, F218, Q268, V306; D10, N167, F168, S174, D179, A182, A183, K186, A187, F218; F17, I59, N167, F168, S174, D179, A182, A183, K186, A187, F218; V28, N167, F168, S174, D179, A182, A183, K186, A187, F218; H39, N167, F168, S174, D179, A182, A183, K186, A187, F218; E47, G50, N167, F168, S174, D179, A182, A183, K186, A187, F218; P57, V58, I59, N167, F168, S174, D179, A182, A183, K186, A187, F218; A63, S65, N167, F168, S174, D179, A182, A183, K186, A187, F218; F69, N167, F168, S174, D179, A182, A183, K186, A187, F218; A126, N167, F168, S174, D179, A182, A183, K186, A187, F218; T139 N167, F168, S174, D179, A182, A183, K186, A187, F218; N167, F168, S174, D179, A182, A183, K186, A187, F218, H225; N167, F168, S174, D179, A182, A183, K186, A187, F218, L230; N167, F168, S174, D179, A182, A183, K186, A187, F218, A245; N167, F168, S174, D179, A182, A183, K186, A187, F218, H251; N167, F168, S174, D179, A182, A183, K186, A187, F218, A281; N167, F168, S174, D179, A182, A183, K186, A187, F218, L287, M289; N167, F168, S174, D179, A182, A183, K186, A187, F218, S293; N167, F168, S174, D179, A182, A183, K186, A187, F218, S304; D10, F17, V28, H39, P57, V58, I59, A63, S65, F69, A126, T139, N167, F168, S174, D179, A182, A183, K186, A187, F218, H225, L230, A281, L287, M289, F293, S304; 5. A variant according to any preceding item, wherein the variant comprises one or more of the following substitutions (e.g., each row of the following table represents a single typical combination of features according to the invention, e.g., of a particular embodiment) variant) or its equivalent amino acid substitution: X167T, X168Y, X174C, X218Y; X167T; X167S; X167A; X167C; X167L; X167T, X174T; X167T, X174C; X155K, X167T, X174C, X175A; X155K, X167T, X174C, X175T; X167S, X174C; X167T, X174C, X218Y; X167T, X168Y, X174C; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X268L, X306I; X10N, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X17Y, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X28T, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X39R, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X47S, X50E, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X57E, X58A, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X63S, X65H, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X69Y, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X126S, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X139P X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y X230R; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X245V; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X251E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X281E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X287T, X289S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X293S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X304R; X10N, X17Y, X28T, X39R, X57E, X58A, X59M, X63S, X65H, X69Y, X126S, X139P, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225, X28, X230 X289S, X293S, X304R; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X202V, X216V, X218Y, X287T, X289S; X155R, X167T, X168Y, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V; X155R, X167T, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y; X155R, X167T, X168Y, X174C, X175V, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X306I; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X287S, X289S, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X287S; 6. The variant according to any one of the preceding paragraphs, wherein the variant comprises one or more of the following substitutions: X167G, A, S, C, U, I, L, V, T; X174G, A, S, C,U,I,L,V,T; X155R,H,K; X175G,A,S,C,U,V,T; X218Y,F,H; and/or X168Y,F,H 7. According to the foregoing The variant of any one, wherein the parent alpha/beta hydrolase, preferably having EC: 3.1.1.-hydrolase activity, is selected from the group consisting of the following peptides: i) with SEQ ID NO: 1 The polypeptide has at least 60% (preferably at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% , or at least 99.5%, or 100%) polypeptides of sequence identity; ii) fragments of the polypeptides of SEQ ID NO: 1, wherein the fragments have alpha/beta hydrolase activity, preferably EC: 3.1.1.-hydrolysis enzymatic activity. 8. The variant according to any preceding item, wherein the parental alpha/beta hydrolase comprises or consists of the polypeptide of SEQ ID NO: 1. 9. The variant according to any of the preceding paragraphs, wherein the variant: i) comprises or consists of a permutation selected from the group consisting of (e.g., the following permutations) Each row of the table represents a single typical combination of features according to the invention, eg, a variant of a particular embodiment): N167T, F168Y, S174C, F218Y; N167T; N167S; N167A; N167C; N167L; N167T, S174T; N167T, S174C; R155K, N167T, S174C, V175A; R155K, N167T, S174C, V175T; N167S, S174C; N167T, S174C, F218Y; N167T, F168Y, S174C; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, Q268L, V306I; D10N, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F17Y, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; V28T, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; H39R, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; E47S, G50E, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; P57E, V58A, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A63S, S65H, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F69Y, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A126S, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; T139P N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y L230R; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A245V; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H251E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A281E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, L287T, M289S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S293S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S304R; D10N, F17Y, V28T, H39R, P57E, V58A, I59M, A63S, S65H, F69Y, A126S, T139P, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225, L28ER, A28ER M289S, F293S, S304R; and/or ii) is selected from the group consisting of SEQ ID NOs: 2-35. 10. A variant that obtains a parent alpha/beta hydrolase (the variant preferably has EC: 3.1.1.-hydrolase activity) and/or improves the stability of the parent alpha/beta hydrolase A method comprising: at positions 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304 introducing substitutions to the parental alpha/beta hydrolase at one or more positions of the variant, wherein the variant has alpha/beta hydrolase activity; and, recovering the variant (e.g., from a lysate, e.g., using an affinity-purified method); preferably, the parent α/β hydrolase is as described in any of the foregoing, further preferably, the parent α/β hydrolase and SEQ ID NO: 1 have at least 95% sequence identity sex. 11. The method according to any preceding item, wherein the variant is at least 71% (preferably: at least 72%, at least 73%, at least 74%, at least 75%) with the polypeptide of SEQ ID NO: 1, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, Polypeptides with at least 98%, at least 98.5%, at least 99%, or at least 99.5%) but less than 100% sequence identity. 12. A polynucleotide encoding the variant of any preceding item. 13. A nucleic acid construct comprising the polynucleotide of any of the foregoing. 14. A performance vector comprising the polynucleotides and/or nucleic acid constructs of any one of the foregoing. 15. A host cell comprising at least one of: i) the variant and/or parental alpha/beta hydrolase according to any one of the foregoing; ii) the polynucleotide according to any one of the foregoing; iii) The nucleic acid construct according to any of the preceding; and/or iv) the expression vector according to any of the preceding. 16. The host cell according to any preceding item, wherein the host cell is a recombinant host cell, preferably an isolated recombinant host cell. 17. The host cell according to any one of the preceding items, wherein the host cell is selected from bacterial cells, yeast cells, mammalian cells, insect cells and plant cells, preferably from bacterial cells and yeast cells. 18. A transgenic plant, transgenic seed or transgenic pollen grain, comprising one or more (preferably multiple) of the following: i) the variant and/or parental alpha/beta hydrolase of any of the foregoing; ii) the foregoing The polynucleotide of any of the foregoing; iii) the nucleic acid construct and/or expression vector of any of the foregoing; and/or iv) the recombinant host cell of any of the foregoing. 19. The transgenic plant, transgenic seed or transgenic pollen grain according to any preceding item capable of producing one or more variants according to any preceding item, preferably for the transgenic plant, transgenic seed or transgenic pollen grain. The transgenic pollen grains are genetically modified to produce one or more of the variants of any of the preceding. 20. The transgenic plant, transgenic seed or transgenic pollen grain according to any preceding one, wherein said transgenic plant, transgenic seed or transgenic pollen grain is homozygous for said (ii) and/or (iii). 21. A method of producing the variant described in any of the foregoing, comprising: (i) culturing the host cell according to any of the foregoing under conditions suitable for the expression of the variant; and (ii) recovering the variant. body. 22. Variants obtained by the method of any of the preceding. 23. Use of the variant and/or parental alpha/beta hydrolase of any preceding item in one or more of: i) degrading zearalenone or a derivative thereof; ii) food, food, intermediate food ; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, Intermediate antidotes; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; pharmaceutical compositions; iii) Manufacture/production of one or more of the following: food , intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); Antidote, Intermediate Antidote; Nutritional Supplement, Intermediate Nutritional Supplement; Prebiotic, Intermediate Prebiotic, Silage Inoculum; Antidote; Veterinary Composition; Pharmaceutical Composition, and/or Mixtures thereof; iv) Green Manufacture/production of stored feed, biogas, bioethanol or sugar, preferably from sugar cane or sugar beet; v) Prevention or treatment of mould poisoning. 24. A composition or a test kit comprising one or more of the following: (i) the variant and/or parental alpha/beta hydrolase of any of the foregoing, preferably, the parental alpha/beta The hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1; (ii) the polynucleotide of any of the foregoing; (iii) the nucleic acid construct of any of the foregoing; (iv) The expression vector of any one of the preceding claims; (v) the host cell of any one of the preceding claims. 25. The composition or kit according to any preceding item, wherein the composition or kit is a veterinary drug composition, pharmaceutical composition or kit. 26. The composition or kit according to any one of the preceding items, further comprising a veterinary drug composition, a pharmaceutically acceptable carrier. 27. The composition or kit according to any preceding item, wherein the composition or kit is a food or feed additive, or a food or feed product. 28. The composition or kit according to any one of the foregoing, further comprising a nutritionally acceptable carrier and/or the parental alpha/beta hydrolase of any one of the foregoing, preferably, the parent α/β hydrolase. The alpha/beta hydrolase has at least 95% sequence identity to the polypeptide of SEQ ID NO: 1. 29. The composition or kit of any of the foregoing, further comprising one or more of the following (for example, as shown in Table 2 herein): capable of removing ochratoxin A (OTA) and/or at least one OTA derivative (eg ochratoxin B and/or ochratoxin C) toxicity (eg modification and/or hydrolysis) and/or binding to ochratoxin A (OTA) and/or at least one OTA derivative (eg ochratoxin B and One or more other polypeptides of ochratoxin C) belonging to the M20 peptidase aminoamylase 1-like protein 2-like aminohydrolase subfamily (eg, M20 peptidase ACY1L2 aminohydrolase) The enzyme subfamily, for example, according to the conserved domain database (eg https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=cd05672) identifier is cd05672, and/or, has amino acid Peptidase activity (eg EC 3.4.11.10), and/or, including carboxypeptidase activity (eg carboxypeptidase A and/or B activity, eg with EC 3.4.17.1 and/or EC 3.4.17.2, respectively), and/or or, thermolysin activity (e.g. with EC 3.4.24.27); (ii) capable of removing one or more mycotoxins (e.g. ZEN) and/or one or more plant- and/or bacterial-toxins (e.g. , Trichothecene mycotoxins such as deoxynivalenol, citrullinol, neosolanisol, mucormycin, flatworm, bacillus A, staphyloxin H, bismuth Fusarium acetate, HT-2 toxin or T-2 toxin; aflatoxins, such as aflatoxins B1, B2, G1 or G2; fumonisins, such as fumonisins B1, B2, B3 or B4 ; polypeptide mycotoxins such as beauvericin or enfusaricin; zearalenone; citrinin; patulin; ergot alkaloids such as ergotamine) and/or one or more plant sources - and One or more other polypeptides of bacterial origin - toxicity of toxins (eg endotoxins, etc.), in particular, said one or more other polypeptides capable of removing one or more other mycotoxins and/or one or more plant origin - and/or Bacterial origin-toxins, for example, fumonisin esterase (eg, as disclosed in WO 2016/134387 A1) and/or zearalenone lactonase (eg, as disclosed in WO 2020/025580 A1) and /or ergopeptidase (eg, as disclosed in WO 2014/056006 A1); (iii) one or more organic adsorbents (eg, live/inactivated/lyophilized/dormant/dead intact Yeast, or yeast-derived products such as yeast cell walls, or yeast oligomers such as mannan) and/or inorganic adsorbents (eg, diatomaceous earth and/or clay minerals such as kaolin or kaolinite, montmorillonite such as montmorillonite , illite, or chlorite; especially, swelling soil); (iv) capable of removing one or more other mycotoxins (e.g., trichothecene mycotoxins such as deoxynivalenol, fenugreek, neosolaniol, mucor Insect toxin, baculosporin A, staphyloxin H, diacetoxyfusaricol, HT-2 toxin or T-2 toxin; aflatoxins, such as aflatoxins B1, B2, G1 or G2 ; fumonisins, such as fumonisins B1, B2, B3 or B4; polypeptide mycotoxins, such as beauvericin or enfusaricin; zearalenone; citrinin; patulin; ergot alkaloids , such as ergotamine) and/or one or more live, inactivated, lyophilized and/or dormant microorganisms that are toxic to one or more plant- and/or bacterial-derived toxins (e.g., endotoxins, etc.) , in particular, the microorganism is a member of the genera Trichospora and Apiotrichum (eg as disclosed in WO 03/053161 A1) or the Rhododendron family (eg as disclosed in EP 3 501 526 A1); (v ) one or more plant products (eg, seaweed, preferably seaweed meal; and/or, algae, preferably algae meal; and/or, thistle, preferably thistle seed; and/or, licorice preparations, preferably licorice powder and/or licorice extracts, eg, as disclosed in WO 2018/121881 A1); (vi) one or more flavouring compounds (eg, plant extracts, eg from oregano, thyme, wintergreen, Coriander, marjoram, peppermint, peppermint, fennel, citrus, lemon, cumin, star anise, clove, cinnamon and/or garlic; and/or, essential oils such as D-limonene, gamma-terpenes, cymene Hydrocarbon, 2-carene, linalool oxide, isomenthone, camphor, linalool, terpinen-4-ol, 2-isopropyl-1-methoxy-4-toluene, L -Menthol, Ethylamine, Alpha-Terpineol, Beta-Caryophyllene, D-Carvone, Methyl Salicylate, Alpha-Caryophyllene, Lavender Acetate, Caryophyllene Oxide, Eugenol Thymol and/ or carvacrol); (vii) one or more vitamins (eg, vitamins A, D, E, K, C, B1, B2, B3, B4, B5, B6, B7, B8, B9 and/or B12; especially is vitamin E). 30. The variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, host cell, transgenic plant, transgenic seed, transgenic pollen grain, composition or kit of any of the preceding Use as medicine (for example, for veterinary use and/or for pharmaceutical use, for example for animals and/or humans) and/or for use in therapy, preferably for the prevention or treatment of diseases, further preferably, the pro- The generation alpha/beta hydrolase has at least 95% sequence identity to SEQ ID NO: 1. 31. Variants, parental alpha/beta hydrolases, polynucleotides, nucleic acid constructs, expression vectors, host cells, transgenic plants, transgenic seeds, transgenic pollen grains, compositions or kits for the prevention or treatment of mycotoxicosis , preferably hormonal disorders induced by zearalenone or its derivatives. 32. A method of preventing or treating mycotoxicosis, the method comprising: administering to a subject at risk or in need one or more of the following: the variant of any of the preceding, parent alpha/beta hydrolase, Polynucleotides, nucleic acid constructs, expression vectors, host cells, transgenic plants, transgenic seeds, transgenic pollen grains, compositions or kits. 33. The method or use of any of the foregoing, wherein the variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, host cell, transgenic plant, Transgenic seeds, transgenic pollen grains, compositions or kits. 34. A food, intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feedstuffs) intermediate additive); antidote, intermediate antidote; nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, silage inoculum; antidote; veterinary composition; pharmaceutical composition, and/or mixtures thereof , which include one or more of the following: a variant of any of the preceding, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, host cell, transgenic plant, transgenic seed, transgenic pollen grain , composition or kit, preferably, the parent α/β hydrolase has at least 95% sequence identity with the polypeptide of SEQ ID NO: 1. 35. A method of degrading zearalenone or a derivative thereof, comprising: i) providing one or more variants of any of the foregoing, and/or, preferably one or more of any of the foregoing Multiple parental alpha/beta hydrolases, preferably, the parental alpha/beta hydrolases have at least 95% sequence identity with the polypeptide of SEQ ID NO: 1; ii) making one or more variants and/or pro- Substitute contact with zearalenone or a derivative thereof (eg, to form an enzyme-substrate mixture). 36. The method for degrading zearalenone or a derivative thereof according to any one of the preceding items, wherein the variant and/or parent alpha/beta hydrolase is when the substrate concentration is 0.5 ppm and the pH is 5.0 The specific activity of the variant is at least 0.6 µmol/min/mg (eg, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, or at least 1.4), preferably, the specific activity of the variant is at least 1.35 µmol/min/mg (such as 1.39 or 1.57).

The present invention is further illustrated by the following examples, but is not limited to the examples or any particular implementation of the examples.

Embodiments of the present invention

Example 1 : encodes zearalenone - Modification, cloning, expression and zearalenone of cleavage polypeptide polynucleotides - Purification of cleaved polypeptides

The complete zearalenone-cleavage polypeptide gene (including a 6x histidine tag fused to the C-terminus of the polypeptide) was synthesized according to the nucleotide sequence and then incorporated into an expression vector by a public company (eg Twist Bioscience).

Competent E. coli BL21 (DE3) was transformed with an expression vector containing the complete zearalenone-cleavage polypeptide gene. Transformants were screened for antibiotic resistance of the expression vector. Any other suitable host cell can also be used in this work.

To prepare competent E. coli BL21 (DE3), cells are seeded in LB medium (eg, Luria/Miller, pellet, 6673, Carl Roth GmbH+CoKG, 25.0 g/L) and grown overnight at 37.0°C with constant shaking. An overnight culture was inoculated in LB medium as the main culture and grown at 37.0°C with continued shaking until the _OD600 was 0.65. 15.0 min prior to harvesting, the main culture was cooled to 4.0°C, and cells were stored at 4.0°C in all subsequent steps whenever possible or otherwise indicated. Cells were harvested at 4.0°C, 2000.0 x g for 10 minutes. The cell pellet was resuspended using 5.0 ml of TSS buffer (LB medium, 10.0% polyethylene glycol-6000, 5.0% dimethylsulfoxide, 20.0 mM magnesium chloride) per 100.0 ml of primary culture.

For transformation of competent E. coli BL21 (DE3), use 5x KCM buffer (500.0 mM KCl, 150.0 mM CaCl, 250.0 mM MgCl) to dilute the competent cell solution 1:1.25. Provide 5.0 µl of the expression vector containing the intact zearalenone-cleavage polypeptide gene to 50.0 µl of the prepared cell solution and incubate at 4.0°C for 10.0 minutes. Heat shock at 42.0°C for 1.5 minutes, followed by incubation at 4.0°C for 1.0 minutes. Cells were regenerated in 1.0 ml LB medium for 1.5 hours at 37.0°C. Inoculate 100.0 µl of cells in 900.0 µl of LB medium with antibiotics (eg, kanamycin 50.0 µg/ml) and incubate overnight at 37.0°C with constant shaking.

Intact zearalenone-cleaved polypeptide genes were expressed intracellularly from positive transformants using a commercially available autoinduction medium.

Inoculate 500.0 µl of the overnight culture in 9.5 ml of autoinduction medium (eg Novagen Overnight Express ^TM Instant TB Medium, 71491, Sigma- Aldrich Handels GmbH), covered with a sealing film (eg Breathe. Easier sealing film, Z763624, Sigma-Aldrich Handels GmbH) to achieve high gas exchange rates. Express cultures were incubated for 24.0 hours at 37.0°C with constant shaking. Cells were harvested at 2000.0 x g for 10 minutes at room temperature.

Cells harvested from the expression step were lysed by sonication. Zearalenone-cleaved polypeptides were obtained from cleared lysates using 6x histidine-tagged affinity purification. Molecular weight and purity were confirmed by SDS-PAGE analysis. The concentration of zearalenone-cleavage polypeptide was determined by NanoDrop ^™ analysis.

Resuspend the harvested cells in 2.0 ml of binding buffer (20.0 mM sodium phosphate, 500.0 mM sodium chloride, 60.0 mM imidazole, pH 7.4) at 4.0 °C. Treat the suspension on a sonicator until cells are completely lysed at 4.0 °C (eg, Q700-220 + Q4579, amplitude 50.0 - treatment time 8.0 min - pulse 20.0 sec - pause 40.0 sec, Qonica L.L.C).

Lysates were clarified by centrifugation at 2000.0 x g for 15 minutes at 4.0°C. 1.4 ml of the supernatant was purified on a HIS-Spin trap column (eg, HIS-Spin Trap, 28-4013-53, GE Healthcare GmbH) following the manufacturer's protocol. Zearalenone-cleavage peptides were eluted in 400.0 µl of elution buffer (20.0 mM sodium phosphate, 500.0 mM sodium chloride, 500.0 mM imidazole, pH 7.4), which was used to study enzymatic properties.

To confirm the molecular weight and purity of the purified zearalenone-cleavage polypeptide, add loading dye to dilutions of the eluate (e.g., 2x loading dye: 0.125 M Tris/HCl pH 6.8, 20.0% glycerol, 4.0% sodium lauryl sulfate, 0.02% bromophenol blue, 2% β-mercaptoethanol), boiled and gelatinized in 12.0% polyacrylamide (eg, Mini-PROTEAN® Tetra Cell System, 1658004EDU + 12% Mini-PROTEAN® TGX ^TM Precast Protein Gels, 4561043EDU, Bio-Rad Laboratories GmbH) (10× Running Buffer: 0.125 M Tris, 1.25 M Glycine, 0.5 % Sodium Lauryl Sulfate, 200 V, 40 minutes). Bands of peptides were visualized by Coomassie staining and compared to standard peptide markers.

To determine the concentration of purified zearalenone-cleavage polypeptide, measure the absorbance at 280.0 nm (eg, NanoDrop ^™ One, ND-ONE-W, Thermo Fisher Scientific Inc.) and calculate the concentration according to the Lambert-Beer rule.

Enzyme oligomerization degree ( degree of enzyme oligomerisation ) analysis ( Blue-Native-PAGE – BN-PAGE )

Comparison of native molecular weights (oligomeric conformations) between individual purified zearalenone-cleavage polypeptides by Blue-Native-PAGE. The eluate was diluted with a native loading dye (e.g., 2x loading dye: 0.126 M Tris/HCl pH 6.8, 20.0 % glycerol, 4.0% Coomassie Blue G-250) and washed in natural Separation on 4-15% polyacrylamide gels under conditions (e.g. Mini-PROTEAN® Tetra Cell System, 1658004EDU + 4-15 % Mini-PROTEAN® TGX ^™ Precast Protein Gels, 4561083EDU, Bio-Rad Laboratories GmbH) (5x Anode Buffer: 0.125 M Tris, 0.960 M Glycine, 1x Cathode Buffer: Anode Buffer + 0.02 % Coomassie Blue G250; 200 V, 40 min). Peptide bands were visualized by Coomassie staining. The oligomeric state of selected zearalenone-cleaved polypeptides was determined using size exclusion chromatography, SEC-MALS or X-ray crystallography. Homodimers (eg SEQ ID NO. 1) migrate faster on the BN-PAGE gel matrix, while homotetramers (eg SEQ ID NO. 2-35) are retained on the gel to a higher degree matrix. The oligomeric conformations of the zearalenone-cleavage polypeptides were determined in terms of homodimeric and homotetrameric peptides.

Determination of Zearalenone -Cleaved Polypeptide Melting Points ( Tm ): The melting point of purified zearalenone-cleaved polypeptides was determined using thermal shift analysis based on the fluorescence of the dye SYPRO Orange during protein denaturation. While water quenches its fluorescence, binding to unfolded hydrophobic surfaces on denatured proteins causes fluorescence to increase.

The purified purified zearalenone-cleavage polypeptide was diluted to 0.2 mg/ml in Teorell Stenhagen buffer pH 7.5 (Östling and Virtama, Acta Physiologica Scandinavica, 1946, 11:4, 289-293). Sypro Orange 5000X stock solution (Sypro Orange, S5692, Sigma-Aldrich Handels GmbH) was diluted 1:100 in Teorell Stenhagen buffer pH 7.5. In a 96-well PCR-plate sealed with a transparent heat-seal film suitable for fluorescence detection, 15.0 µl of the diluted zearalenone-cleavage peptide was added with 3.0 µl of the diluted Sypro Orange solution and 12.0 µl of Teorell Stenhagen pH 7.5 buffer. Melting curves were measured on a qPCR-cycler using a linearly increasing temperature from 20.0°C to 95.0°C over 20 minutes, with 147 measurement points per measurement curve (460.0 nm extinction, 550.0 nm emission) (e.g. Mastercycler qPCR ep realplex 2s, 6300000604, Eppendorf AG). The melting point is determined at the inflection point of the fluorescence signal (in other words, the temperature at which the rate of increase in the fluorescence signal is highest). Fluorescence intensity plotted against temperature gradient for 147 measurement points. Differentials were calculated for every 147 measurement points as d fluorescence signal/d temperature for two subsequent measurement points. The temperature at which the highest value of dF/dT is calculated is the maximum value of the first derivative, representing the inflection point and thus the melting point of the zearalenone-cleavage polypeptide.

surface 3. Zearalenone - The melting point of the cleaved polypeptide ( Tm ) SEQ ID NO. Amino acid replacement compared to SEQ ID NO: 1 oligomeric conformation Tm (°C) 1 - dimer 50.0 2 N167T, F168Y, S174C, F218Y tetramer 54.0 3 N167T tetramer 53.1 4 N167S tetramer 53.0 5 N167A tetramer 52.2 6 N167C tetramer 52.9 7 N167L tetramer 52.3 8 N167T, S174T tetramer 53.5 9 N167T, S174C tetramer 53.8 10 R155K, N167T, S174C, V175A tetramer 53.9 11 R155K, N167T, S174C, V175T tetramer 53.8 12 N167S, S174C tetramer 54.2 13 N167T, S174C, F218Y tetramer 54.3 14 N167T, F168Y, S174C tetramer 54.3 15 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 65.2 16 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, Q268L, V306I tetramer 63.2 17 D10N, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 64.2 18 F17Y, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 64.2 19 V28T, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 65.8 20 H39R, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 64.7 twenty one E47S, G50E, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 59.6 twenty two P57E, V58A, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 63.2 twenty three A63S, S65H, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 65.3 twenty four F69Y, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 64.2 25 A126S, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 64.7 26 T139P N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y tetramer 67.8 27 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225S tetramer 64.2 28 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y L230R tetramer 69.8 29 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A245V tetramer 62.7 30 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H251E tetramer 66.8 31 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A281E tetramer 65.3 32 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, L287T, M289S tetramer 64.7 33 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S293S tetramer 63.2 34 N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S304R tetramer 64.7 35 D10N, F17Y, V28T, H39R, P57E, V58A, I59M, A63S, S65H, F69Y, A126S, T139P, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225, L28ER, A28ER M289S, F293S, S304R tetramer 65.8

Determination of specific activity

Prepare Zearalenone Degradation Assay Buffer (118.5 mM NaCl, 8.55 mM acetic acid, 14.9 mM acetate, 0.1 mg/ml BSA, pH 5.0, (Jantratid, E., Janssen, N., Reppas, C. & Dressman) , J. B. (2008): Dissolution media simulating conditions in the proximal human gastrointestinal tract: An update. Pharmaceutical Research 25(7): 1663-1576), containing 0.1 mg/ml bovine serum albumin and 0.52 ppm zearalenone as Substrate), pre-warmed at 37.0°C, and transfer 960 µl of assay buffer to a reaction tube in a 96-deep-well plate. The plate was sealed with a removable lid and stored in a DWP-thermal shaker at 37.0°C until the start of the zearalenone degradation assay.

Use purified zearalenone-cleavage polypeptides by diluting in sample buffer (Teorell Stenhagen buffer pH 7.5, containing 0.1 mg/ml bovine serum albumin) to concentrations higher than those analyzed in the final zearalenone degradation experiments 25 times higher to prepare the enzyme working solution, the concentration of zearalenone-cleaving polypeptide can effectively degrade zearalenone under the given conditions.

To begin the zearalenone degradation experiment, add 40.0 µl of the enzyme working solution to a tube containing 960.0 µl of assay buffer to achieve a final ZEN concentration of 0.5 ppm in the assay reaction. The addition of pH 7.5 enzyme working solution did not alter the pH 5.0 zearalenone degradation reaction.

The zearalenone degradation reaction was incubated at 37.0°C with constant shaking in a DWP-thermal shaker. Immediately after the zearalenone degradation reaction begins, resuspend and mix with a pipette tip, transfer 120.0 µl of the 0.0 h sample to a tube on a new PCR plate, and seal with a cap. Additional samples were taken from the zearalenone degradation reaction after several time points (eg, 5.0, 10.0, 20.0, 30.0, 45.0, 60.0, 90.0 min). Once the sample is removed from the degradation reaction, thermally inactivate zearalenone cleavage in the sample by incubating it in a thermo-block (eg, Thermal Shake lite, 460-029, VWR) for 10.0 minutes at 99.0°C peptide. Subsequently, the tubes were centrifuged (3.0 min, room temperature, 2500 x g) and 90.0 µl of the supernatant was transferred to a rimmed PCR plate (eg, twin.tec® PCR plate, 0030128648, Eppendorf AG), which was sealed with a heat-sealed film (for example, heat-sealing film, 0030127838, Eppendof AG) to seal. These sample plates were stored at 4.0°C until HPLC-FLD measurement. Zearalenone concentrations were measured according to a modified HPLC-FLD method described by Vekiru et al. (Vekiru et al. (2016) 'Isolation and characterization of enzymatic zearalenone hydrolysis reaction products' World Mycotoxin Journal 9:353-363).

Analysis was performed on HPLC-FLD with extinction at 278.0 nm and emission at 465.0 nm. The retention time for zearalenone was 1.8 minutes when separated on a Kinetex 5µm EVO C18 100 A 50.0 x 2.1 mm column (00B-4633-AN, Phenomenex Inc.) at 40.0°C using the following method : Using solvent A (95.0 % acetonitrile + 4.9 % water + 0.1 % formic acid) and solvent B (5.0 % acetonitrile + 94.9 % water + 0.1 % formic acid), gradient: 0.0-0.5 min 10 % phase B, 0.5-2.0 min linear Increase to 50.0 % Phase B, then reduce to 10 % Phase B in 0.1 min, continuing for a total run time of 2.3 min. The flow rate was set to 1.5 ml/min and the injection volume was 5.0 μl. Similar acquisition settings were used for the various analyses.

Zearalenone was quantified based on the calibration of the zearalenone external standard. The zearalenone concentration (µM) of the reaction samples taken is plotted against the sampling time point. The slope of linear zearalenone degradation over time was calculated as μmol zearalenone decreased per minute in the volume of the enzymatic reaction. Taking into account possible dilutions of purified zearalenone-cleavage polypeptides and including these appropriate dilution factors in the calculation, calculate in µmol zearalenone degraded per minute per mg of purified enzyme (µmol ZEN/min). /mg) expressed the specific enzymatic activity (specific enzymatic activity).

surface 4 : pH5.0 specific enzyme activity Enzyme identification Specific activity (µmol ZEN/min/mg) PRIOR ART COMPARISON SEQUENCE 1 0.00 PRIOR ART COMPARISON SEQUENCE 2 0.00 SEQ ID NO. 1 of the present invention 1.57 SEQ ID NO. 28 of the present invention 1.39

Those skilled in the art will readily appreciate that the present invention is adapted to carry out the objects and obtain the objects and advantages as well as those inherent therein. Furthermore, it will be apparent to those skilled in the art that various substitutions and modifications of the invention disclosed herein can be made without departing from the scope and spirit of the invention. The compositions, methods, processes, treatments, molecules, and specific compounds described herein are presently representative of certain embodiments, are exemplary, and are not intended to limit the scope of the invention. Variations and other uses encompassed within the spirit of the invention to those skilled in the art will be defined by the scope of the claims. The listing or discussion of a previously published document in this specification is not necessarily an admission that the document is part of the state of the art or is common general knowledge.

The invention exemplarily described herein may suitably be practiced without any element or element, limitation or limitation, the invention not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be read in an expansive rather than a restrictive sense. Furthermore, the terms and expressions used herein have been used as terms of description rather than of limitation and, where such terms and expressions are used, are not intended to exclude any equivalents of the features shown and described, or parts thereof, however, it should be recognized that , various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed by the exemplary embodiments and optional features, those skilled in the art may resort to modifications and variations of the invention embodied herein, and that such modifications and variations are It is considered to be within the scope of the present invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgroups falling within the general disclosure also forms part of the invention. This includes a general description of the invention, with limitations or negative limitations that exclude any subject matter from the derivation, whether or not the ex vivo material is expressly recited herein. All documents, including patent applications and scientific publications, are hereby incorporated by reference in their entirety.

Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described in terms of the Markush group, those skilled in the art will appreciate that the invention is thus also described in terms of any individual member or subgroup of members of the Markush group.

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Claims (18)

A variant of a parental alpha/beta hydrolase comprising substitutions at one or more positions corresponding to SEQ ID NO: 1 (preferably using the numbering of SEQ ID NO: 1) 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304, wherein the variant has alpha/beta hydrolase activity, and wherein the variant is with the polypeptide of SEQ ID NO: 1 having at least 71% (preferably: at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%) but less than 100% sequence identity, than Preferably, the variant has hydrolase EC: 3.1.1.-activity. . A variant according to any one of the preceding claims, wherein the variant has one or more of the following properties: i) improved properties relative to the parental alpha/beta hydrolase, wherein the improved properties comprise increased properties The temperature stability is preferably compared to the improved temperature stability of SEQ ID NO: 1; the variant can be tetramerized, preferably has a tetrameric quaternary structure, further preferably, the variant Tetramerization is homotetramerization; ii) a melting point (Tm) greater than 50°C, preferably the Tm is in the range of about 51°C to about 70°C; iii) capable of degrading zearalenone and / or derivatives thereof; iv) comprising at least one (preferably at least two, more preferably at least 80% (preferably at least 83%, more preferably at least 90%) sequence identity with amino acid sequences selected from the group consisting of: Preferably at least three, most preferably at least four) amino acid motifs: a) SEQ ID NO: 36 (QVDLGEX ₁ X ₂ MN), wherein X is V or _I , preferably V, and wherein X ₂ is V or T, preferably V; b) SEQ ID NO: 37 (EYDPEWX ₃ RX ₄ FX ₅ EGTV), wherein X ₃ is G or A, preferably A, and wherein X ₄ is A or V, preferably Preferably A, and wherein X ₅ is F or Y; c) SEQ ID NO: 38 (MLX ₆ QVKTPX ₇ LX ₈ THH), wherein X ₆ is S or G, preferably S, and wherein X ₇ is I or V, preferably I, and wherein _X8 is I or L, preferably I; d) SEQ ID NO: 39 (PAX ₉ LLX ₁₀ PEQTGSWWSYEX ₁₁ X ₁₂ X ₁₃ GLLX ₁₄ EX ₁₅ FHVX ₁₆ AVDX ₁₇ RGQGRSX ₁₈ WTPX ₁₉ RYSLDNFGNDLVRFIX ₂₀ LVX ₂₁ KRPVX ₂₂ VX ₂₃ GNSSGGX ₂₄ LAAWLSAYX ₂₅ MPGQX ₂₆ RX ₂₇ X ₂₈ LCEDX ₂₉ X ₃₀ FFASELVPAX ₃₁ GHSVX ₃₂ QX ₃₃ AGPX ₃₄ FELX ₃₅ R) where X ₉ is V or L, preferably L, and where X is ₁₀ is L or I, preferably L, and wherein _X is P or E, preferably P, and wherein _X is V or A, preferably V, and wherein _X is I or M, preferably I, and wherein _X is A or S, preferably _A , and wherein _X is S, N or H, preferably S, and wherein X is F or Y, preferably F, and wherein X ₁₇ is I or L, preferably I, and wherein X ₁₈ is T or S, preferably T, and wherein X ₁₉ is K or R, preferably K, and wherein X ₂₀ is S, A or N, preferably S, and wherein _X is V or I, preferably V, and wherein _X is I or V, preferably I, and wherein _X is S or A, Preferably S, and wherein X is V or L, preferably V, and wherein _X is _A or S, preferably A, and wherein _X is I or L, preferably I, and wherein X ₂₇ is A or G, preferably A, and wherein _X is A or V, preferably A, and wherein _X is T, A or P, preferably T, and wherein _X is P or A, Preferably P, and wherein _X is Y or H, preferably H, and wherein _X is L or R, preferably R, and wherein _X is A or G, preferably A, and wherein X ₃₄ is A or V, preferably A, and wherein X ₃₅ is Y or F, preferably Y. A variant according to any one of the preceding claims, wherein the variant comprises one or more of the following substitutions, or an amino acid substitution equivalent thereto: X167T, X168Y, X174C, X218Y; X167T; X167S; X167A; X167C; X167L; X167T, X174T; X167T, X174C; X155K, X167T, X174C, X175A; X155K, X167T, X174C, X175T; X167S, X174C; X167T, X174C, X218Y; X167T, X168Y, X174C; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X268L, X306I; X10N, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X17Y, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X28T, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X39R, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X47S, X50E, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X57E, X58A, X59M, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X63S, X65H, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X69Y, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X126S, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X139P X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y X230R; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X245V; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X251E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X281E; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X287T, X289S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X293S; X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X304R; X10N, X17Y, X28T, X39R, X57E, X58A, X59M, X63S, X65H, X69Y, X126S, X139P, X167T, X168Y, X174C, X179E, X182C, X183R, X186R, X187R, X218Y, X225, X28, X230 X289S, X293S, X304R; and / or X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X202V, X216V, X218Y, X287T, X289S; X155R, X167T, X168Y, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V; X155R, X167T, X174C, X175V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y; X155R, X167T, X168Y, X174C, X175V, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X189H, X190S, X192V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X306I; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X287S, X289S; X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X268L, X287S, X289S, X306I; X34V, X100N, X140A, X155R, X167T, X168Y, X174C, X175V, X179E, X182C, X183R, X186R, X187R, X202V, X218Y, X225N, X266R, X268L, X269Q, X287S; or its equivalent amino acid replacement. The variant according to any one of the preceding claims, wherein the parental alpha/beta hydrolase, preferably having EC: 3.1.1.-hydrolase activity, is selected from the group consisting of the following peptides: i) with the polypeptide of SEQ ID NO: 1 at least 60% (preferably at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98% , at least 98.5%, at least 99% or at least 99.5% or 100%) sequence identity; ii) a fragment of the polypeptide of SEQ ID NO: 1, wherein the fragment has alpha/beta hydrolase activity, preferably EC: 3.1.1.-hydrolase activity. A variant according to any preceding claim, wherein the variant: i) comprises or consists of a permutation selected from the group consisting of: N167T, F168Y, S174C, F218Y; N167T; N167S; N167A; N167C; N167L; N167T, S174T; N167T, S174C; R155K, N167T, S174C, V175A; R155K, N167T, S174C, V175T; N167S, S174C; N167T, S174C, F218Y; N167T, F168Y, S174C; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, Q268L, V306I; D10N, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F17Y, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; V28T, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; H39R, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; E47S, G50E, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; P57E, V58A, I59M, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A63S, S65H, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; F69Y, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; A126S, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; T139P N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y L230R; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A245V; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H251E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, A281E; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, L287T, M289S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S293S; N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, S304R; D10N, F17Y, V28T, H39R, P57E, V58A, I59M, A63S, S65H, F69Y, A126S, T139P, N167T, F168Y, S174C, D179E, A182C, A183R, K186R, A187R, F218Y, H225, L28ER, A28ER M289S, F293S, S304R; and/or ii) is selected from the group consisting of SEQ ID NOs: 2-35. A method for obtaining a variant of a parental alpha/beta hydrolase, preferably having EC: 3.1.1.-hydrolase activity, and/or increasing the stability of said parental alpha/beta hydrolase, The method comprises: at positions 167, 168, 174, 218, 155, 175, 179, 182, 183, 186, 187, 268, SEQ ID NO: 1 (preferably using the numbering of SEQ ID NO: 1), One of 306, 10, 17, 28, 39, 47, 50, 57, 58, 59, 63, 65, 69, 126, 139, 225, 230, 245, 251, 281, 287, 289, 293 and 304 introducing substitutions into the parental alpha/beta hydrolase at or multiple positions, wherein the variant has alpha/beta hydrolase activity; and recovering the variant; preferably, the parental alpha/beta The hydrolase of any preceding claim, further preferably, the parental alpha/beta hydrolase has at least 95% sequence identity to SEQ ID NO: 1. A polynucleotide encoding the variant of any of the preceding claims. A nucleic acid construct comprising the polynucleotide of any one of the preceding claims. An expression vector comprising the polynucleotide and/or nucleic acid construct of any one of the preceding claims. A recombinant host cell, comprising at least one of the following: i) the variant and/or parental alpha/beta hydrolase of any one of the preceding claims; ii) the polynucleotide of any one of the preceding claims; iii) the nucleic acid construct described in any one of the preceding claims; and/or iv) The performance carrier of any one of the preceding claims. A transgenic plant, transgenic seed or transgenic pollen grain, comprising one or more (preferably multiple) of the following: i) the variant and/or parental alpha/beta hydrolase of any one of the preceding claims; ii) the polynucleotide of any one of the preceding claims; iii) the nucleic acid construct described in any one of the preceding claims; iv) a performance carrier according to any of the preceding claims; and/or v) The recombinant host cell of any of the preceding claims. A food, intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives ); antidote, intermediate antidote; nutritional supplement, intermediate nutritional supplement; prebiotic, intermediate prebiotic, silage inoculum; antidote; veterinary composition; pharmaceutical composition, and/or mixtures thereof, which Including one or more of the following: i) the variant and/or parental alpha/beta hydrolase of any one of the preceding claims; preferably, the parental alpha/beta hydrolase has at least 95% of the polypeptide of SEQ ID NO: 1 sequence identity; ii) the polynucleotide of any one of the preceding claims; iii) the nucleic acid construct described in any one of the preceding claims; iv) the performance carrier described in any one of the preceding claims; v) the recombinant host cell of any one of the preceding claims; vi) The transgenic plant, transgenic seed and/or transgenic pollen grain of any of the preceding claims. A composition or kit comprising one or more of the following: i) the variant and/or parental alpha/beta hydrolase of any one of the preceding claims, preferably, the parental alpha/beta hydrolase has at least 95% of the polypeptide of SEQ ID NO: 1 sequence identity; ii) the polynucleotide of any one of the preceding claims; iii) the nucleic acid construct described in any one of the preceding claims; iv) the performance carrier described in any one of the preceding claims; v) the recombinant host cell of any one of the preceding claims; vi) the transgenic plant, transgenic seed or transgenic pollen grain of any one of the preceding claims; and/or vii) the food and intermediate food according to any one of the preceding claims; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, Food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculation bacteria; antidote; veterinary composition; Pharmaceutical compositions, and/or mixtures thereof. The composition or kit according to any one of the preceding claims, further comprising a nutritionally acceptable carrier and/or the parental alpha/beta hydrolase of any one of the preceding claims, preferably, The parental alpha/beta hydrolase has at least 95% sequence identity to the polypeptide of SEQ ID NO: 1. The variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, recombinant host cell, transgenic plant, transgenic seed, transgenic pollen grain, food product, intermediate food of any one of the preceding claims ; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, Intermediate antidotes; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; pharmaceutical compositions and/or mixtures thereof, compositions and/or kits , for use as medicine and/or for treatment, preferably, for the prevention or treatment of disease, further preferably, the parent α/β hydrolase and the polypeptide of SEQ ID NO: 1 have at least 95% sequence identity. The variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, recombinant host cell, transgenic plant, transgenic seed, transgenic pollen grain, food product, intermediate food of any one of the preceding claims ; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, Intermediate antidotes; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; pharmaceutical compositions and/or mixtures thereof, compositions and/or kits , for the prevention or treatment of fungal poisoning, preferably, for the prevention or treatment of hormonal disorders caused by zearalenone or its derivatives. The variant, parental alpha/beta hydrolase, polynucleotide, nucleic acid construct, expression vector, recombinant host cell, transgenic plant, transgenic seed, transgenic pollen grain, food product, intermediate food of any one of the preceding claims ; Forage, intermediate forage; Feed, intermediate feed; Additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or feed intermediate additives); Antidote, Intermediate antidotes; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; use of pharmaceutical compositions and/or mixtures thereof in one or more of the following : i) degradation of zearalenone and/or its derivatives; ii) food, food, intermediate food; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, food intermediate additives, forage intermediate additives or Feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculation bacteria; antidote; veterinary compositions; pharmaceutical compositions; iii) Manufacture/production of one or more of the following: food, intermediate food; forage, intermediate forage; feed, intermediate feed; additive (preferably, food additive, forage additive or feed additive), intermediate additive (preferably, food intermediate additives, intermediate additives for forages or intermediate additives for feed); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculants; antidotes; veterinary compositions; pharmaceuticals compositions and/or mixtures thereof; iv) Manufacture/production of silage, biogas, bioethanol or sugar, preferably from sugar cane or sugar beet; v) Prevention or treatment of mold poisoning. A method for degrading zearalenone and/or derivatives thereof, comprising: a) provide one or more of the following: i) the variant and/or parental alpha/beta hydrolase of any one of the preceding claims, preferably, the parental alpha/beta hydrolase has at least 95% of the polypeptide of SEQ ID NO: 1 sequence identity; ii) the recombinant host cell described in any one of the preceding claims; iii) the transgenic plant of any one of the preceding claims; iv) The food and intermediate food according to any one of the preceding claims; forage, intermediate forage; feed, intermediate feed; additives (preferably, food additives, forage additives or feed additives), intermediate additives (preferably, Food intermediate additives, forage intermediate additives or feed intermediate additives); antidote, intermediate antidote; nutritional supplements, intermediate nutritional supplements; prebiotics, intermediate prebiotics, silage inoculation bacteria; antidote; veterinary composition; Pharmaceutical compositions and/or mixtures thereof; and/or v) the composition or kit of any one of the preceding claims; b) contacting one or more substances from (a) with zearalenone and/or a derivative thereof.